Journal: OncoTargets and therapy

Article Title: Overexpression of BUB1B contributes to progression of prostate cancer and predicts poor outcome in patients with prostate cancer

doi: 10.2147/OTT.S101994

Figure Lengend Snippet: The expression of BUB1B in cell lines. Notes: ( A ) The relative expression of the mRNA level using qRT-PCR. * P <0.05. ( B ) The protein expression level by Western blot. Abbreviations: BUB1B, BUB1 mitotic checkpoint serine/threonine kinase B; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Article Snippet: The membranes were blocked with 10% skim milk in Tris-buffered saline–Tween 20 and probed with anti-BUB1B antibody (1:1,000, #4116, Cell Signaling Technology) or anti-β-actin antibody (1:2,000, sc-47778, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: Overexpression of BUB1B contributes to progression of prostate cancer and predicts poor outcome in patients with prostate cancer

doi: 10.2147/OTT.S101994

Figure Lengend Snippet: Depletion of BUB1B by siRNA has no effect on the proliferation and migration of DU145 cells. Notes: ( A and B ) qRT-PCR and Western blot analysis for BUB1B expression after siRNA transfection. ( C ) The growth curve of DU145 cells. ( D and E ) Wound-healing assay. ( F and G ) Transwell analysis to detect cell migration. Abbreviations: BUB1B, BUB1 mitotic checkpoint serine/threonine kinase B; h, hours; qRT-PCR, quantitative reverse transcription polymerase chain reaction; siBUB1B, small interfering RNA oligonucleotides for inhibiting BUB1B; siNC, small interfering RNA mock oligonucleotides; siRNA, small interfering RNA.

Article Snippet: The membranes were blocked with 10% skim milk in Tris-buffered saline–Tween 20 and probed with anti-BUB1B antibody (1:1,000, #4116, Cell Signaling Technology) or anti-β-actin antibody (1:2,000, sc-47778, Santa Cruz, Dallas, TX, USA).

Techniques: Migration, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Wound Healing Assay, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA

Journal: OncoTargets and therapy

Article Title: Overexpression of BUB1B contributes to progression of prostate cancer and predicts poor outcome in patients with prostate cancer

doi: 10.2147/OTT.S101994

Figure Lengend Snippet: Overexpression of BUB1B promotes the proliferation and migration of DU145 cells. Notes: ( A and B ) qRT-PCR and Western blot analysis for BUB1B expression. ( C ) The growth curve of DU145 cells. ( D and E ) Wound-healing assay. ( F and G ) Transwell analysis to detect cell migration. * P <0.05. Abbreviations: BUB1B, BUB1 mitotic checkpoint serine/threonine kinase B; h, hours; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Article Snippet: The membranes were blocked with 10% skim milk in Tris-buffered saline–Tween 20 and probed with anti-BUB1B antibody (1:1,000, #4116, Cell Signaling Technology) or anti-β-actin antibody (1:2,000, sc-47778, Santa Cruz, Dallas, TX, USA).

Techniques: Over Expression, Migration, Quantitative RT-PCR, Western Blot, Expressing, Wound Healing Assay, Negative Control, Reverse Transcription, Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: Overexpression of BUB1B contributes to progression of prostate cancer and predicts poor outcome in patients with prostate cancer

doi: 10.2147/OTT.S101994

Figure Lengend Snippet: Immunohistochemical staining for BUB1B expression in PCa and adjacent noncancerous tissues. Notes: ( A ) The whole scanned picture of the tissue array. ( B ) The percentage of strong (green column) and weak BUB1B staining (gray column) in different groups by the level of Gleason score (<7, =7, and >7). * P <0.05. ( C ) Immunostaining of benign and cancer tissues. Magnification of the upper panel is ×50; the bottom panel is ×200. ( D ) Statistical analyses showing higher IRS in cancerous tissues than in adjacent noncancerous tissues (IRS: PCa =4.18±1.83 vs benign =3.53±1.43, P =0.011). Abbreviations: BUB1B, BUB1 mitotic checkpoint serine/threonine kinase B; IRS, immunoreactivity score; PCa, prostate cancer.

Article Snippet: The membranes were blocked with 10% skim milk in Tris-buffered saline–Tween 20 and probed with anti-BUB1B antibody (1:1,000, #4116, Cell Signaling Technology) or anti-β-actin antibody (1:2,000, sc-47778, Santa Cruz, Dallas, TX, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, Immunostaining

Journal: OncoTargets and therapy

Article Title: Overexpression of BUB1B contributes to progression of prostate cancer and predicts poor outcome in patients with prostate cancer

doi: 10.2147/OTT.S101994

Figure Lengend Snippet: Association of BUB1B expression with the clinicopathological characteristics of PCa

Article Snippet: The membranes were blocked with 10% skim milk in Tris-buffered saline–Tween 20 and probed with anti-BUB1B antibody (1:1,000, #4116, Cell Signaling Technology) or anti-β-actin antibody (1:2,000, sc-47778, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Overexpression of BUB1B contributes to progression of prostate cancer and predicts poor outcome in patients with prostate cancer

doi: 10.2147/OTT.S101994

Figure Lengend Snippet: High BUB1B expression is linked to poor prognosis in patients with PCa. Notes: ( A ) Kaplan–Meier analysis of the biochemical recurrence-free time of PCa patients with high BUB1B expression levels was shorter than those with low BUB1B expression levels. ( B ) The overall survival time of patients with PCa was not correlated to BUB1B expression levels. Abbreviations: BUB1B, BUB1 mitotic checkpoint serine/threonine kinase B; PCa, prostate cancer.

Article Snippet: The membranes were blocked with 10% skim milk in Tris-buffered saline–Tween 20 and probed with anti-BUB1B antibody (1:1,000, #4116, Cell Signaling Technology) or anti-β-actin antibody (1:2,000, sc-47778, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Overexpression of BUB1B contributes to progression of prostate cancer and predicts poor outcome in patients with prostate cancer

doi: 10.2147/OTT.S101994

Figure Lengend Snippet: Prognostic value of BUB1B expression for the biochemical recurrence-free survival in univariate and multivariate analyses by Cox regression

Article Snippet: The membranes were blocked with 10% skim milk in Tris-buffered saline–Tween 20 and probed with anti-BUB1B antibody (1:1,000, #4116, Cell Signaling Technology) or anti-β-actin antibody (1:2,000, sc-47778, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) Disease Free Survival or Overall Survival was evaluated in mCRPC patients with high BUB1B expression (top quartile) compared to patients with low BUB1B expression (bottom quartile) from (Cancer Cell, 2010) and SU2C RNA-seq data sets (downloaded from cBioportal) (log-rank test, ** p < 0.01, *** p < 0.001). (b) BUB1B mRNA expression was compared between normal or PC samples or between different disease stages in TCGA-PRAD (RNAseq data downloaded from http://xena.ucsc.edu/ ) MSKCC (Cancer Cell, 2010 from cBioportal), GSE94767, GSE32269, (microarray data from https://www.ncbi.nlm.nih.gov/gds ) (student’s t test, **** p < 0.0001). BUB1B protein was evaluated by IHC in (c) three pairs of LuCaP androgen dependent (AD) and castration resistant (CR) patient derived xenografts (PDXs) (LuCaP35, LuCaP77 and LuCaP96) or (d) in TMAs from metastatic CRPC samples from 10 patients with 16 bone and 17 visceral tissue (liver, spleen, lung and lymph node) (n=33) and benign tissue (n=2). Graphs represent the quantification of BUB1B positive cells IHC for (c) LuCaP PDXs and (d) TMAs. (student’s t test, * p < 0.05, ** p < 0.01). BUB1B (e) mRNA and (f) protein levels were evaluated in RWPE-1 [non-tumorigenic (NT), grey], LNCaP, VCaP (ADPC, yellow), C4-2B, 22Rv1, CWR-R1, PC3 and DU145 (CRPC, orange) cells. Graphs represent the average ± SEM of two or three independent experiments performed in triplicate.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Expressing, RNA Sequencing, Microarray, Derivative Assay

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) Cells excluding trypan blue were counted following transduction with shBUB1B (3’UTR) or control (shGFP) in LNCaP (AR positive) ADPC cells, C4-2B (AR positive), 22Rv1 (AR/ AR-V7 positive) and PC3 (AR-negative) CRPC cells. Graphs represent the average of four or five independent experiments performed in quadruplicate ± SEM. The area under the curve was calculated for each cell line (Mann-Whitney test, * p < 0.05, *** p < 0.001). (b) CDK1 and cyclin A levels after BUB1B depletion in 22Rv1 by Western blot. Western blot quantification is shown in Supplementary Figure 1 b. (c) Cell cycle distribution was measured after BUB1B depletion (vs control shGFP) in 22Rv1 cells by flow cytometry of propidium iodide (PI) stained cells. Graphs represent distribution of the cell population across the cell cycle of the average ± SEM of four independent experiments (Mann-Whitney test, * p < 0.05). (d) Representative images of mitosis duration evaluated by live cell imaging using the Incucyte Zoom System in 22Rv1 cells after BUB1B depletion (vs control shGFP). Graphs show quantification of one representative experiment (50 randomly chosen cells per group), of two independent experiments (unpaired t-test, * p < 0.05).

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Transduction, Control, MANN-WHITNEY, Western Blot, Flow Cytometry, Staining, Live Cell Imaging

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) BUB1B levels were determined by western blot of lysates from LNCaP and VCaP ADPC cells ectopically expressing BUB1B or EV. (b) LNCaP and VCaP BUB1B vs EV cell proliferation was measured by trypan blue exclusion in the absence of androgens after eight days. Graphs represent the average ± SEM of three or four independent experiments performed in triplicate (Mann-Whitney test, * p < 0.05). (c) Colony formation was evaluated in LNCaP EV and BUB1B in soft agar assays after fifteen days. Images are representative of one of three independent experiments. Graphs represent the average of three independent experiments performed in triplicate (unpaired t-test, **** p < 0.0001). (d) LNCaP EV or BUB1B expressing cells were subcutaneously xenografted in castrated SCID mice (n=10 for EV and n=29 for BUB1B) and tumor volumes (mm 3 ) were measured by caliper every 3 days for 85 days. Tumor volume over time was evaluated using a mixed model with cubic effect. Differences were observed between groups at the end of the experiment (day 85). (e) End point PSA in plasma was determined by ELISA (Mann-Whitney test, **** p < 0.0001) (f) Images of end-stage tumors from LNCaP EV and BUB1B xenografts (scale bar = 1 cm). (g) BUB1B was evaluated by western blot in tumor tissue extracts. Tumor images and the corresponding tumor samples were ordered based on tumor size. (h) Graph shows the correlation between tumor weight and BUB1B expression (Pearson correlation, ** p < 0.01).

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Western Blot, Expressing, MANN-WHITNEY, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) LNCaP and VCaP ADPC cells expressing BUB1B (WT), D882N, K795R (kinase dead mutants) or control cells (EV) were grown in the absence of androgen and BUB1B protein was evaluated by western blot. Growth of LNCaP and VCaP expressing EV, BUB1B (WT), D882N and K795R was determined by trypan blue after eight days in culture. Graphs represent the average of three independent experiments performed in triplicate. (Kruskal-Wallis test, * p < 0.05, ** p < 0.01). (b) Colony formation of LNCaP EV, BUB1B, D882N and K795R was evaluated by soft agar assays after 3 weeks. Images are representative of three independent experiments (one-way ANOVA, * p < 0.05, **** p < 0.0001). BUB1B, D882N,K795R or EV control cells were expressed in 22Rv1 CRPC. (c) 22Rv1 cell proliferation was measured by trypan blue exclusion assay after BUB1B knockdown (siRNA against non-coding sequences) and ectopic expression of BUB1B, D882N,K795R or EV control. The graph represents the average of four independent experiments performed in triplicate (unpaired t-test, **** p < 0.0001). The lower panel shows BUB1B protein by western blotting in 22Rv1 EV, BUB1B, D882N and K795R expressing cells after knockdown of endogenous BUB1B using siBUB1B RNA targeting the 3’UTR.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Expressing, Control, Western Blot, Trypan Blue Exclusion Assay, Knockdown

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) AR was evaluated by western blot in LNCaP EV and BUB1B cells growing in castrated conditions (2% CSS) at 0, 4 and 8 days. (b) AR was knocked down in LNCaP EV or BUB1B cells by siRNA and cell number was quantified by trypan blue exclusion assay after eight days. Graph represents three independent experiments performed in triplicate (Mann-Whitney test, * p < 0.05). AR levels after AR knockdown using siRNA in LNCaP EV and BUB1B cells are shown.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Western Blot, Trypan Blue Exclusion Assay, MANN-WHITNEY, Knockdown

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: (a) Cell proliferation determined by trypan blue exclusion after eight days of enzalutamide treatment (10 µM) in LNCaP and VCaP EV and BUB1B cells. The graph represents three independent experiments done in triplicate (Mann-Whitney test, * p < 0.05). (b) Colony formation in soft agar was measured after fifteen days. Images are representative of three independent experiments performed in triplicate. Graphs are representative of three to four independent experiments performed in triplicate (one-way ANOVA, ** p < 0.01, **** p < 0.0001). (c) BUB1B was evaluated by western blot in LNCaP xenograft derived T_EV and T_BUB1B cells. (d) T_EV and T_BUB1B cells were grown in the absence of androgens and proliferation was measured by trypan blue exclusion assays. The graph represents three independent experiments performed in triplicates. The area under the curve was calculated (unpaired t-test, * p < 0.05). (e) Proliferation of T_EV and T_BUB1B cells treated with 10 µM enzalutamide (or vehicle) for eight days is shown. The graph represents three independent experiments performed in triplicate (Kruskal-Wallis test, * p < 0.05). (f) LNCaP EV or T_BUB1B expressing cells were subcutaneously xenografted into castrated SCID mice (EV n=10 and T_BUB1B n=29) and tumor volumes (mm3) were measured by caliper every 3 days. Once tumors reach 200 mm3, animals were split into two groups and treated daily with enzalutamide (n=9) or vehicle (n=11). Graph shows tumor volume over time. (g) PSA levels in plasma were determined by ELISA (Mann-Whitney test, **** p < 0.0001). (h) Images of end-stage tumors from T_BUB1B vehicle (n=11) or enzalutamide (n=9) xenografts are shown. (i) BUB1B protein was evaluated in tumors by western blot. Tumor images and protein samples were ordered based on tumor size, vehicle tumors #1 and 2 were repeated in both blots for comparison.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: MANN-WHITNEY, Western Blot, Derivative Assay, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: BUB1B MITOTIC KINASE DRIVES THERAPY RESISTANT PROSTATE CANCER

doi: 10.1101/2025.11.09.687448

Figure Lengend Snippet: Cell proliferation was measured after GSK-923295 (CENP-E inhibitor) treatment (0, 0.03, 0.06, 0.12 and 0.25 µM) for 7 days in (a) LNCaP EV and BUB1B cells or (b) LNCaP T_EV and T_BUB1B cells. Graph represents four independent experiments performed in triplicate (one-way ANOVA, * p < 0.05, ** p < 0.01). (c) BUB1B cells growing in castration conditions were treated with GSK-923295 (10-30 nM), enzalutamide (10-30 µM) or the combination for 7 days and cell number was evaluated by trypan blue exclusion assay. Two independent experiments were performed in triplicate. Combinatory index (CI) was calculated using the CompuSyn software. A CI < 1 indicates synergism, = 1 indicates additivity,> 1 indicates antagonism.

Article Snippet: For overexpression experiments, pCMV-6 BUB1B wild type was purchased from Origene (RC205844), and pLAP-BUB1B D882B (114062) and K795R (114060) were purchased from Addgene.

Techniques: Trypan Blue Exclusion Assay, Software

Journal: STAR Protocols

Article Title: Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors

doi: 10.1016/j.xpro.2024.103355

Figure Lengend Snippet:

Article Snippet: pLVX-hSyn-Gαi1-YFP-IRES∗-KB1753-Nluc (also named hSyn-Gαi1 ONE-GO) , Janicot et al. & Addgene , Cat #204333.

Techniques: Recombinant, Saline, Luciferase, Plasmid Preparation, Software

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Acetylation of BubR1 at K250 identifies unattached kinetochores. ( A ) Immunofluorescent staining of metaphase chromosome spreads. Mitotic HeLa cells were enriched by treatment with colcemid (100 ng/ml) for 18 h, followed by a 5-min release, and then subjected to co-immunostaining using anti-BubR1 and anti-acetylated BubR1 (AcK250) monoclonal antibodies (mAbs). Kinetochores were categorized into three groups based on anti-AcK250 mAb immunostaining: those immunostained at both sister kinetochores (Paired); one kinetochore (Single); or neither (No). Scale bar, 5 μm. ( B ) Proportion of anti-AcK250 mAb immunostaining patterns in panel (A). Results are from two independent experiments (mean ± s.e.m.; n = 40 chromosomes). P- values were obtained using a t -test. ( C ) Representative images of microtubule attachment at kinetochores using SIM. Mitotic HeLa cells were treated with 10 μM MG132 to enrich the metaphase population, fixed, and then co-immunostained using anti-α-tubulin, anti-AcK250, and CREST antibodies. CREST-marked kinetochores were divided into three groups based on 3D-reconstructed images: unattached kinetochore (No attachment), monotelic kinetochore (Single attachment), and amphitelic kinetochore (Bi-attachment). Scale bar, 5 μm (top). Bottom, 3D-reconstructed images of anti-AcK250 mAb staining. Scale bar, 0.5 μm. The SIM images were acquired using an AP DeltaVision OMX Ultra High-Resolution Fluorescence Microscope (GE Healthcare). Image reconstruction and alignment were performed using SoftWoRx software. Three-dimensional reconstruction was performed using ImageJ software (3D projection). ( D ) Relative intensity of anti-AcK250 immunostaining normalized to CREST. Number of kinetochores scored: No attachment (No), n = 20; monotelic attachment (Single), n = 22; bipolar attachment (Bi), n = 16. Numbers of kinetochores scored are from 30 different cells, each from two independent experiments. Statistical differences between groups were assessed using the Mann–Whitney U test; P -values are indicated. (E–H) HeLa cells treated with 200 ng/ml nocodazole (Noc), 2 μM paclitaxel/taxol (Tax), 100 μM monastrol (Mon), or 10 μM MG132, respectively, were co-immunostained with CREST, anti-α-tubulin, and anti-AcK250 ( E, F ) or anti-BubR1 ( G, H ). Scale bar, 5 μm. ( F, H ) Graphs are from two independent experiments (mean ± s.e.m.). Number of cells scored for (F): Noc, n = 7; Tax, n = 15; Mon, n = 21; MG312, n = 8 and for (H): Noc, n = 28; Tax, n = 17; Mon, n = 33; MG132, n = 12.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Staining, Immunostaining, Bioprocessing, Fluorescence, Microscopy, Software, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Lysine 250 acetylation of BubR1 (BubR1–AcK250) is associated with kinetochore expansion. ( A ) Representative images of M-phase HeLa cells immunostained with anti-BubR1, CREST, and α-tubulin antibodies. Cells were treated with 10 μM MG132 to enrich the metaphase population. Kinetochores were categorized into three groups based on 3D-reconstructed SIM images: No attachment, Single attachment, or Bi-attachment. Scale bar, 5 μm. Bottom, 3D reconstruction. Scale bar, 0.5 μm. ( B ) Volume of BubR1 in cells exhibiting no attachment (No), monotelic attachment (Single), and amphitelic attachment (Bi). Number of kinetochores scored: No, n = 131; Single, n = 89; Bi, n = 115. Results are from 155 cells in three independent experiments (mean ± s.e.m.). ( C ) Representative SIM images of FLAG-tagged BubR1-WT, -K250R, and -K250Q in mitotic kinetochores. HeLa-FRT-TO cells expressing a single copy of FLAG-BubR1, -K250R , or -K250Q were depleted of endogenous BubR1 via siRNA transfection . Simultaneously, doxycycline was used to induce the expression of BubR1-WT, -K250R , and -K250Q , respectively. Cells were treated with nocodazole (Noc) alone or with MG132 (Noc + MG132) to assess the effect without perturbing the stability of K250R. Cells were treated with nocodazole (200 ng/ml, 20 h) alone or in combination with MG132 (10 μM, final 2 h). Scale bar, 5 μm. ( D ) Volume (μm 3 ) of FLAG (left) and relative intensities of FLAG to CENP-A (right) in nocodazole-treated cells with or without MG132. Number of kinetochores scored in nocodazole-only treatment: WT, n = 12; K250R, n = 14; and K250Q, n = 14. For nocodazole + MG132 treatment: WT, n = 72; K250R, n = 72; and K250Q, n = 72. The graphs are from two independent experiments (mean ± s.e.m.).

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Expressing, Transfection

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: K250-BubR1 acetylation is involved in the maintenance of crescent-shaped fibrous corona. ( A ) Representative images of immunostaining with anti-MAD2 and anti-ZW10 antibodies after induction of BubR1-WT, -K250R , and -K250Q expression in HeLa-FRT-TO cells. Nocodazole (200 ng/ml) alone or with MG132 was applied before fixation. Endogenous BubR1 was depleted via siRNA. Scale bar, 5 μm. ( B ) Representative SIM images of immunostained ZW10 at kinetochores of HeLa cells expressing FLAG-tagged BubR1-WT, -K250R , or -K250Q following double thymidine block and release. FRT/TO HeLa cells were treated with doxycycline for 55 h to induce expression of ectopic BubR1 variants. Endogenous BubR1 was depleted using siRNA targeting the 3′ UTR. Cells were fixed for immunostaining 10 h after the final thymidine release to enrich the mitotic population. ( C ) Effects of BubR1 acetylation status on CENP-E and RZZ complex formation. Cells were treated with nocodazole alone (+Noc) or with MG132 (+Noc + MG132). Relative protein levels were measured using a densitometer, and the ratios are indicated at the bottom. ( D ) Representative images showing immunostained ZW10 and anti-CENP-A antibodies in FRT/TO HeLa cells expressing BubR1-WT, K250R , or K250Q with (siCtrl) or without CENP-E (siCENP-E). Cells were transfected with siRNA targeting luciferase (siCtrl) or CENP-E (siCENP-E) for 48 h prior to fixation. Nocodazole was administered for 20 h before analysis.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Immunostaining, Expressing, Blocking Assay, Transfection, Luciferase

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Aurora kinase B activity is required for BubR1 acetylation and maintenance of fibrous corona. ( A ) Immunostaining with anti-AcK250 mAb after treatment of nocodazole-arrested cells with the indicated mitotic kinase inhibitors: 10 nM of BI 2536 (PLK1 inhibitor), 2 μM of Reversine (MPS1 inhibitor), 200 nM of Hesperadin (Aurora B inhibitor), and 2 μM of ZM447439 (Aurora B inhibitor). Kinase inhibitors were applied during the final 2 h of a 20-h nocodazole treatment. Scale bar, 5 μm. ( B ) Relative intensities of AcK250 at kinetochores from panel (A): Nocodazole-arrested cells without kinase inhibitor treatment (NT), and nocodazole + BI 2536-treated (BI 2536), reversine-treated (Rev), hesperadin-treated (Hes), or ZM447439-treated (ZM) cells (mean ± s.e.m.). Each dot represents the relative AcK250 signal intensity per cell. Number of cells scored: NT, n = 31; BI, n = 44; Rev, n = 18; Hes, n = 21; ZM, n = 28. Results are from two independent experiments. ( C ) Assessing the effect of Aurora B kinase activity in MCC maintenance. HeLa cells were transfected with Myc-tagged Aurora B-WT or kinase-dead mutant K106R -expressing constructs. Endogenous Aurora B was depleted using siRNA targeting the 3′ UTR. BI 2536 (10 nM), Reversine (2 μM), Hesperadin (200 nM), or ZM447439 (2 μM) were added during the final 2 h of a 20-h nocodazole treatment (left). Immunoprecipitation was performed using an anti-CDC27 (APC3) antibody, followed by WB with indicated antibodies. To assess MCC stability, MG132 was added with the indicated kinase inhibitors in nocodazole-arrested cells (right). WB with indicated antibodies in total cell lysate (TCL, 5%) is shown at the bottom. WB with 9E10 shows the expression of Myc-tagged AurkB-WT or AurkB-K106R . Note the high level of AcK250–BubR1 in WT-AurkB -expressing mitotic cells, but not in AurkB-K106R -expressers (AcK250) or interphase cells (lane 2). Both the BubR1 level in anti-CDC27 immunoprecipitate and AcK250 BubR1 in TCL are high in nocodazole-treated mitotic cells, which are inhibited by AurkB inhibitors but not by BI 2536 or Reversine (lanes 4–7). Phospho-AurkB is detected only in WT-AurkB -expressers (AurkB-pT232). ( D ) Representative images of anti-AcK250 immunostaining in cells expressing wild-type Aurora B ( AurkB-WT ) or kinase-dead Aurora B ( AurkB-K106R ). Scale bar, 5 μm. ( E ) Intensity of anti-AcK250 immunofluorescence measured from panel (D). Number of cells counted: WT, n = 11; AurkB-K106R, n = 7 (mean ± s.e.m.). Each point represents the mean AcK250 signal intensity per cell. ( F ) Effect of AurkB activity in the expansion of BubR1 (left) and ZW10 (right) at kinetochores in response to nocodazole treatment. Scale bar, 5 μm. ( G ) Volume of BubR1 and ZW10 at the kinetochores from panel (F). Number of kinetochores: BubR1 in WT, n = 105; BubR1 in K106R, n = 121; ZW10 in WT, n = 153; ZW10 in K106R, n = 85 (mean ± s.e.m.). The results are from 131 cells in three independent experiments. ( H ) Interaction between BubR1, Aurora B, and phosphorylated Aurora B (AurkB-pT232) was assessed by IP with anti-BubR1 and WB with indicated antibodies. Mitotic HeLa cells were collected by mitotic shake-off after 20 h of nocodazole treatment (M phase, M). Interphase-attached cells (Attached, A) were employed for control.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Activity Assay, Immunostaining, Transfection, Mutagenesis, Expressing, Construct, Immunoprecipitation, Immunofluorescence, Control

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Aurora B phosphorylates serine 16 and serine 39 of BubR1 upon nocodazole treatment, which is required for K250 acetylation. ( A ) Identification of Aurora B-mediated phosphorylation sites of BubR1 in response to nocodazole treatment. (Top) Schematic illustration of BubR1 marked with AurkB-binding region and phosphorylation sites. Potential phosphorylation sites (Ser16 and Ser39) by AurkB and K250 acetylation site are marked with red. N-terminus of BubR1 (1–150) binds to AurkB (results from Fig. ). (Bottom) IP-WB analysis to identify phosphorylation sites that crosstalk with K250 acetylation. mCherry-tagged BubR1 expression constructs were mutagenized in vitro to substitute the phosphorylation sites with alanine, based on proteome analysis . They were then transfected into HeLa cells and treated with nocodazole (200 ng/ml) for 20 h. Mitotic cells were collected by shake-off, and attached cells (Attach) were employed as a control. Mitotic cell lysates, along with lysates from attached cells (WT, Attach), were subjected to immunoprecipitation with anti-mCherry antibody, followed by WB with anti-AcK250 mAb. The same blot was reprobed with anti-BubR1 and anti-mCherry antibodies for normalization. Total cell lysates were subjected to WB with anti-phospho-H3 and anti-cyclin B antibodies to assess the mitosis stage. WB with anti-mCherry and anti-β-actin antibodies in TCL were used as loading controls. Relative band intensities (AcK250/BubR1) were measured using a densitometer and are indicated. ( B ) Identification of the Aurora B-binding region in BubR1. IP with 9E10 (anti-Myc) and WB with anti-AurkB (Aurora B kinase) were performed. All BubR1-expressing constructs were Myc-tagged. The same blot was reprobed with 9E10 for normalization. Two percent of TCL was subjected to WB with the indicated antibodies. ( C ) Effect of Ser16 or Ser39 phosphorylation in K250 acetylation. Immunofluorescence assay in cells expressing the indicated mCherry-tagged BubR1 variants. Nocodazole-treated cells were formaldehyde-fixed and subjected to immunostaining with anti-AcK250, anti-ACA (CREST), and anti-mCherry antibodies. ( D ) Graph showing the intensities of anti-AcK250 immunofluorescence from panel (C). Anti-AcK250 immunofluorescence signals were normalized to anti-mCherry. The results are from two independent experiments. Number of cells: WT, n = 30; K250R, n = 20; K250Q, n = 24; S16A, n = 28; S39A, n = 29 (mean ± s.e.m.). ( E ) WB analysis showing the effects of mitotic kinase inhibitors on phosphorylation of BubR1 (1–150) in unattached kinetochore (nocodazole treatment). HeLa cells were subjected to nocodazole treatment and mitotic shake-off. The mitotic lysates were then resuspended in in vitro phosphorylation buffer containing 100 μM ATP (see the “Materials and methods” section). MBP-tagged recombinant BubR1 (amino acids 1–150) was employed as the substrate. The mitotic lysate and recombinant BubR1 were incubated in the buffer for 2 h with/without indicated inhibitors (10 nM BI 2536, 2 μM reversine, 200 nM hesperadin, and 2 μM ZM447439). In parallel, the lysates were subjected to WB with anti-AurkB-pT232, -AurkB, and -β-actin. ( F ) In vitro phosphorylation assay showing that phosphorylation of S39 and S16 is AurkB-dependent. Recombinant BubR1 (amino acids 1–150) was incubated with anti-AurkB immunoprecipitate of nocodazole-arrested HeLa cells. Reactions were carried out for 2 h in the presence or absence of the AurkB inhibitor ZM447439 (2 μM). [γ- 32 P]-ATP label detects the phosphorylation of the substrate BubR1 (1–150). WB with anti-MBP and anti-AurkB in the same lysate was performed for control.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Construct, In Vitro, Transfection, Control, Immunoprecipitation, Immunofluorescence, Immunostaining, Recombinant, Incubation

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Phosphorylation of Ser39 (and Ser16) by AurkB is required for K250 acetylation in SAC signaling. ( A ) IP-WB analysis to assess the effect of Ser16 and Ser39 phosphorylation in MCC maintenance upon nocodazole treatment. The indicated mCherry-tagged BubR1 variants were transfected, and endogenous BubR1 was depleted by co-transfection of siRNA for BubR1 targeting the 3′ UTR. Immunoprecipitation was performed with an anti-mCherry antibody, followed by WB with the indicated antibodies. Results for nocodazole-arrested HeLa cells treated with MG132 for 2 h before harvest are shown at right (+Noc + MG132). WB of 2% TCL is shown at the bottom. (B, C) Effects of phosphorylation and acetylation in MCC maintenance. ( B ) Effects of AurkB-mediated phosphorylation at serines 39 (and Ser16) in MCC maintenance. Phospho-deficient mutants S16A-BubR1 and S39A-BubR1 , as well as the S16A;S39A double mutant, and phospho-deficient mutations introduced into the acetylation-mimetic form ( S16A;K250Q, S39A;K250Q , and S16A;S39A;K250Q ) were transfected into HeLa cells, along with siBubR1 to deplete endogenous BubR1. The cells were treated with nocodazole and subjected to IP with anti-mCherry antibody, followed by WB with the indicated antibodies. ( C ) Effect of the phospho-mimetic S16D-BubR1 or S39D-BubR1 on MCC maintenance. The acetylation-deficient K250R mutation was introduced into S16D or S39D ( S16D;K250R and S39D;K250R ) variants, and their capabilities for maintaining MCC after nocodazole treatment were assessed. All BubR1 variants were tagged with mCherry, transfected into HeLa cells, and subjected to IP and WB using the indicated antibodies. ( D ) Effect of AurkB-mediated phosphorylation and K250 acetylation on mitotic timing. Endogenous BubR1 was depleted using siRNA, and the indicated mCherry-tagged BubR1 variants were transfected into HeLa cells stably expressing H2B-GFP . Images were captured at 5-min intervals. Arrows mark anaphase onset.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Phospho-proteomics, Transfection, Cotransfection, Immunoprecipitation, Mutagenesis, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: AurkB-AcK250–BubR1 pathway coordinates the unattachment signal to the maintenance of fibrous corona and SAC. (A, B) Effects of AurkB-mediated phosphorylation and subsequent K250 acetylation in fibrous corona expansion. ( A ) SIM images of anti-Bub1 or anti-MAD2 immunostaining in nocodazole-treated cells are shown. The indicated BubR1 constructs were transfected with siBubR1 targeting the 3′ UTR 48 h before fixation, and nocodazole (200 ng/ml) was applied for 20 h. Volumes were measured from the immunofluorescence assays shown. Number of cells scored: Bub1 in WT, n = 63; Bub1 in S16A, n = 64; Bub1 in S39A, n = 70; MAD2 in WT, n = 196; MAD2 in S16A, n = 196; MAD2 in S39A, n = 69 (mean ± s.e.m.). ( B ) SIM images of anti-ZW10 immunostaining in nocodazole-treated cells. Endogenous BubR1 was depleted via co-transfection with siBubR1. Nocodazole (200 ng/ml) was administered for 20 h, two days post transfection. Insets show enlarged images of ZW10 and CREST staining. Scale bar, 5 μm. ( C ) Volume of ZW10 measured from the immunofluorescence assay shown in panel (B). Number of cells scored: WT, n = 27; S16A, n = 29; S39A, n = 27; S16A;K250Q, n = 31; S39A;K250Q, n = 32; K250Q, n = 29; S16D, n = 31; S39D, n = 32; S16D;K250R, n = 27; S39D;K250R, n = 27; K250R, n = 27 (mean ± s.e.m.). ( D ) Model illustrating the coordination of AurkB-mediated phosphorylation and K250 acetylation in spindle checkpoint signaling. At unattached kinetochores, AurkB phosphorylates BubR1 at S39 (and S16 as well), promoting BubR1 acetylation at K250 by PCAF. This acetylation ensures the maintenance of the RZZ complex and MCC. It also sustains CENP-E at the kinetochore, preparing lateral to end-on attachment (left). Upon successful end-on capture, PP2A activity recruited by AcK250–BubR1 antagonizes AurkB activity, stabilizing KT-MT attachment. Simultaneously, BubR1 immediately deacetylates and degrades through APC/C-mediated ubiquitination, resulting in MCC disassembly and SAC silencing.

Article Snippet: Phosphorylation of BubR1 was detected by immunoblotting using a phospho-serine antibody (AP0932; ABclonal) when radio-labeling was avoided.

Techniques: Phospho-proteomics, Immunostaining, Construct, Transfection, Immunofluorescence, Cotransfection, Staining, Activity Assay, Ubiquitin Proteomics

Journal: Developmental cell

Article Title: Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1.

doi: 10.1016/j.devcel.2007.09.005

Figure Lengend Snippet: Figure 1. AF15q14/Blinkin Interacts with Bubs (A) Immunoprecipitation of FLAG-tagged hMis12. Cultures of HeLa that stably expressed FLAG-tagged hMis12 were used: asynchronous (AS), double thymidine block (DTB), and nocodazole-arrested (Noc). Control HeLa did not contain FLAG-tagged hMis12. Immunoprecipitates (IP) were assayed by antibodies against FLAG, Bub1, and BubR1. (B) Yeast 2-hybrid interactions. (See text for details.) Plasmids pGBD and pGAD carried DNA-binding and activating domains of yeast GAL4, respec- tively. For control, p53 and SV40 T antigen were used. (C) Two-hybrid interactions between one of BLKN, BLKM, or BLKC and hMis12-interacting proteins were examined. (D) Positive 3-hybrid interaction of BLKC with hMis13-hMis14. (E) Immunoblot of HeLa using anti-blinkin antibody. (F) The blinkin band shifted in nocodazole-blocked (Noc) extracts (left). Following nocodazole block and release (B&R), the band of blinkin returned to the lower position (right). (G) Antibodies against blinkin and Bub1 were used for immunostaining of HeLa cells. Cells at prometaphase (top) and metaphase (bottom) are shown. The inset is the enlarged merged images. Bar, 10 mm. (H) Constructs made for blinkin, Bub1, and BubR1 (top). The TPR motif is in red. Blue region is conserved in yeast Mad3. TPR of human (h), mouse (m), S. pombe (sp), and S. cerevisiae (sc) (bottom). Identical and similar residues are boxed and hatched, respectively. TPR consensus (red) and knob-hole association (blue) are also shown. Red arrows indicate residues altered in substitution mutants. (I) Two-hybrid interactions were examined between blinkin fragments or Bub3 and full-length Bub1 or the fragments as indicated. (J and K) Two-hybrid interactions were examined between BLKN and Bubs mutants.

Article Snippet: The cDNA gene for BubR1 (TC119395) was purchased from OriGene, Inc.

Techniques: Immunoprecipitation, Stable Transfection, Blocking Assay, Control, Binding Assay, Western Blot, Immunostaining, Construct

Journal: Developmental cell

Article Title: Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1.

doi: 10.1016/j.devcel.2007.09.005

Figure Lengend Snippet: Figure 2. Blinkin RNAi Abolishes Bubs’ Kinetochore Signals and Spindle Checkpoint Function (A) Immunoblot of blinkin in HeLa extracts after RNAi for 0–72 hr (top). Control immunoblot after luciferase RNAi is also shown. The loading control was tubulin. HeLa cells were fixed 24 hr after blinkin or control RNAi (bottom). DNA and blinkin were observed, respectively, by Hoechst and anti-blinkin antibody. (B) RNAi was performed for blinkin, Bub1, BubR1, and luciferase. Cells were fixed after 24 hr, and stained for DNA, blinkin, Bub1, and BubR1. (See text for details.) (C) Immunoblot of hMis12, blinkin, Bub1, BubR1, and tubulin (loading control) in the RNAi extracts of luciferase, hMis12, blinkin, Bub1, and BubR1 after 24 and 72 hr (left). Localization of hMis12 and blinkin was diminished after hMis12 RNAi, but not after luciferase RNAi (right). (D) Frequency of cells that showed accelerated mitosis and abnormal, nonsegregated chromosomes in four different RNAi cells.

Article Snippet: The cDNA gene for BubR1 (TC119395) was purchased from OriGene, Inc.

Techniques: Western Blot, Control, Luciferase, Staining

Journal: Developmental cell

Article Title: Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1.

doi: 10.1016/j.devcel.2007.09.005

Figure Lengend Snippet: Figure 4. BubR1 TPR Mutants Fail to Suppress BubR1 RNAi (A) Schematized experimental procedures. (B) Plasmid carrying RNAi-resistant BubR1 was made by site-directed mutagenesis. (C) Immunoblot of BubR1, GFP, and tubulin in transfected HeLa cells. The band position of RNAi-resistant GFP-BubR1 detected by anti-BubR1 an- tibody was higher than that of endogenous BubR1. GFP-BubR1204-1050 was detected by anti-GFP antibody, but not by anti-BubR1 antibody because this anti-BubR1 antibody recognizes the amino-terminal region of BubR1 (Taylor et al., 2001). (D) Time-lapse micrographs of HeLa cells that expressed the wild-type or mutant BubR1 protein (RNAi resistant) after BubR1 RNAi. Bar, 10 mm. (E) The duration from NEBD to anaphase onset was measured in a number of movies, and the time distributions are plotted.

Article Snippet: The cDNA gene for BubR1 (TC119395) was purchased from OriGene, Inc.

Techniques: Plasmid Preparation, Mutagenesis, Western Blot, Transfection

Journal: Developmental cell

Article Title: Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1.

doi: 10.1016/j.devcel.2007.09.005

Figure Lengend Snippet: Figure 5. Bub1 TPR Mutant Fails to Suppress Bub1 RNAi Cells (A) Schematized experimental procedures (left). Plasmid carrying RNAi-resistant Bub1 was made by site-directed mutagenesis (right). (B) Immunoblot of Bub1, GFP, and tubulin in transfected HeLa cells. The position of bands for the RNAi-resistant GFP-Bub1 detected by anti-Bub1 antibody was higher than that for endogenous Bub1. (C and D) GFP signals (left, only GFP; middle, GFP-Bub1 WT; right, GFP-Bub1 L122G) expressed in HeLa cells were observed after DNA (bottom, magenta) and tubulin (green) staining. Misaligned chromosomes or micronuclei are indicated by the arrows. The proportion (%) of mitotic cells containing misaligned chromosomes (white column) and interphase cells that showed micronuclei or abnormally large nuclei (black column) were measured after Bub1 RNAi (right). (E) Simultaneous depletion of BubR1 and Bub1 or BubR1 and Bub3 by RNAi was performed after GFP-BubR1 F175G (RNAi-resistant) transfection. DNA and GFP-BubR1 F175G were observed. Bars, 10 mm. (F) Immunoblot of BubR1, Bub1, Bub3, and tubulin. (G) The yeast 2-hybrid interactions were examined between the Bub1 and BubR1 WT or BubR1 F175G mutant.

Article Snippet: The cDNA gene for BubR1 (TC119395) was purchased from OriGene, Inc.

Techniques: Mutagenesis, Plasmid Preparation, Western Blot, Transfection, Staining

Journal: Developmental cell

Article Title: Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1.

doi: 10.1016/j.devcel.2007.09.005

Figure Lengend Snippet: Figure 7. BLKM+C Partly Restores Kinetochore Localization of Bub1 and BubR1 in Blinkin RNAi Cells (A–C) HeLa cells were treated according to the procedures depicted in Figure 6C, using plasmids and siRNAs indicated. Cells were fixed and stained with Hoechst 33342, antibodies against Zwint-1(A), Bub1(B), or BubR1(C). Cross-section images were deconvolved and stacked (Toyoda and Yanagida, 2006). (D) A summary cartoon for the interaction linkage map of kinetochore and checkpoint proteins (left). Functionally distinct Bub1 and BubR1 are simultaneously controlled by blinkin through an interaction between the common TPR motif of Bubs and blinkin (right; see text for details).

Article Snippet: The cDNA gene for BubR1 (TC119395) was purchased from OriGene, Inc.

Techniques: Staining

Journal: Nature communications

Article Title: Lack of Diaph3 relaxes the spindle checkpoint causing the loss of neural progenitors.

doi: 10.1038/ncomms13509

Figure Lengend Snippet: Figure 5 | Lack of Diaph3 disables the SAC. (a–d) Immunostaining for BubR1 (green) and pHH3 (red) of cortical sections from WT (a) and Diaph3 ko (c) E10.5 embryos. (b,d) Enlargements of the boxed areas in a,c, respectively. BubR1 (a hallmark of SAC activation) accumulated more in normal than in Diaph3-deficient cells (quantification in e; n ¼ 1,473 control cells from 3 animals and 1,144 ko cells from 3 animals; Po0.0001, z-test). (f) The density of mitotic cells in the cortex of E10.5 embryos was lower in the ko than in WT (n ¼ 48 WT and 49 ko of 100 mm wide cortical stripes from 3 animals each genotype; Po0.0001; Student’s t-test). (g) Quantification of postmetaphasic cells in the population of mitotic cells. The percentage of mitotic cells that underwent the metaphase–anaphase transition was higher in the ko than in control littermates (n ¼ 786 control cells from 6 animals and 889 ko cells from 5 animals; Po0.0001, z-test). (h,i) Reduction of BubR1 protein levels in the mutant telencephalon detected by western blotting and quantified relatively to tubulin. The same membranes were blotted for Diaph3 to confirm the absence of the protein (h). The level of BubR1 decreased by half in the ko (i; n ¼ 15 embryos in 4 pools for each genotype). (j) Western blotting (WB) detects BubR1 on IP with anti-Diaph3 antibodies from TG cortical lysates. Diaph3 was used as a positive control for the IP (Input). No signal was found in the eluted fraction (flow-through, FT) in presence of Diaph3 antibodies. Detection of the protein in the lysate (Input) required overexposure of the film. Scale bars, 50 mm (a,c) and 5 mm (b,d). Error bars represent s.e.m.

Article Snippet: The second was obtained following the same strategy, but after insertion of the mouse BubR1 ORF (Origene) upstream of the IRES-EGFP.

Techniques: Immunostaining, Activation Assay, Control, Mutagenesis, Western Blot, Positive Control

Journal: Nature communications

Article Title: Lack of Diaph3 relaxes the spindle checkpoint causing the loss of neural progenitors.

doi: 10.1038/ncomms13509

Figure Lengend Snippet: Figure 7 | Working model of Diaph3 function in the spindle checkpoint. (a) Molecular interactions between Diaph3, CPC and SAC proteins during cell division. Documented interactions are depicted in red and new findings are shown in purple. Diaph3 localizes with CPC proteins and co-immunoprecipitates with Survivin and BubR1. (b) Absence of Diaph3 probably disrupts the Diaph3–APC–Eb1 complex, which interacts with and stabilizes the CPC complex at the spindle–kinetochore interface. Loss of Diaph3 impairs the localization of CPC proteins and the ability of dividing cells to activate the SAC. Diaph3-deficient cells fail to accumulate BubR1, leading to slippage to anaphase and mitotic catastrophe and/or cell death of aneuploid progeny.

Article Snippet: The second was obtained following the same strategy, but after insertion of the mouse BubR1 ORF (Origene) upstream of the IRES-EGFP.

Techniques: